WO2011135692A1 - Virtual western blotting system - Google Patents

Virtual western blotting system Download PDF

Info

Publication number
WO2011135692A1
WO2011135692A1 PCT/JP2010/057569 JP2010057569W WO2011135692A1 WO 2011135692 A1 WO2011135692 A1 WO 2011135692A1 JP 2010057569 W JP2010057569 W JP 2010057569W WO 2011135692 A1 WO2011135692 A1 WO 2011135692A1
Authority
WO
WIPO (PCT)
Prior art keywords
substance
molecular weight
antibody
information
binding information
Prior art date
Application number
PCT/JP2010/057569
Other languages
French (fr)
Japanese (ja)
Inventor
喜好 ▲高▼山
正裕 関根
Original Assignee
株式会社エヌビィー健康研究所
埼玉県
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社エヌビィー健康研究所, 埼玉県 filed Critical 株式会社エヌビィー健康研究所
Priority to PCT/JP2010/057569 priority Critical patent/WO2011135692A1/en
Priority to JP2012512589A priority patent/JP5685777B2/en
Publication of WO2011135692A1 publication Critical patent/WO2011135692A1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0426—Bulk waves, e.g. quartz crystal microbalance, torsional waves

Definitions

  • the present invention relates to a microanalysis method and a microanalysis apparatus for a substance. More specifically, the present invention relates to a microanalysis method and microanalysis apparatus for biological materials, particularly proteins.
  • proteins are known to have various functions.
  • protein has been recognized as a source of biological tissue material as one of the three major nutrients, but in recent years, including peptides and amino acids of degradation products, blood pressure lowering action, endocrine function, immunity, and nervous system
  • physiologically active functions such as action and antioxidant action is increasing.
  • proteins and their degradation products are listed as one of the causes of cancer, hypertension, dementia and the like, and are also a causative agent of allergies, and thus are attracting attention in the fields of health and medicine.
  • proteins separated by electrophoresis are detected using an antigen-antibody reaction with high detection sensitivity.
  • This method is particularly useful in the field of life science because it can not only detect but also determine the binding state and modification state of proteins.
  • the Western blotting method still has some problems such as complicated operation, measurement time, and compatibility of quantitative data.
  • electrophoresis is used for protein separation, and immunostaining is used for detection of separated protein. Electrophoresis is superior in protein resolution, but because immunostaining uses antibodies that are proteins, it is difficult to react with the antigen and the target protein in the gel to develop color, and it is necessary to transfer it to a thin film once. There is.
  • the conventional Western blotting method when a protein is detected with an antibody, it takes about half a day to a day and considerable experience and knowledge about biochemical experiments until the final detection result is confirmed. Is required. Furthermore, the conventional method has problems in quantitativeness and reproducibility, and a quick and simple technique having information equivalent to that of the Western blotting method is required.
  • a method for detecting a substance present in a trace amount in a sample as quickly and simply as possible with an antibody a method using a crystal resonator (for example, Patent Documents 1 to 6), a method using surface plasmon resonance (for example, Patent Documents 7 and 8), electrochemical measurement methods (for example, Patent Documents 13 and 14), and the like are known.
  • the crystal resonator is a passive element that causes oscillation with high frequency accuracy by the piezoelectric effect of crystal.
  • the frequency changes in proportion to its weight. Therefore, a trace component is detected using this frequency change.
  • an object of the present invention is to provide a method for identifying a substance that binds to an arbitrary antibody, and to detect a substance associated with the antibody in relation to its molecular weight.
  • Another object of the present invention is to provide an identification processing method for a substance that binds to an arbitrary antibody, which detects the reactivity with an antibody in association with its molecular weight.
  • the present invention is an identification processing method for a substance that binds to an arbitrary antibody, (A) acquiring molecular weight information of the target substance and molecular weight information acquisition time; (B) acquiring binding information between the substance and the antibody, and antibody binding information acquisition time; (C) associating molecular weight information of the substance with antibody binding information from the information obtained in steps (a) and (b); (D) displaying the molecular weight information and antibody binding information associated in step (c); Is an identification processing method.
  • an apparatus used for separating a substance in a sample based on the molecular weight, and detecting the molecular weight information of the substance and binding information with an arbitrary antibody in association with each other,
  • A means for obtaining molecular weight information of each separated substance and time from obtaining a sample to obtaining each molecular weight information;
  • B a means for acquiring binding information of each separated substance with an antibody and a time from sample introduction to acquisition of each antibody binding information;
  • C means for associating molecular weight information of each substance and antibody binding information from the information obtained by means (a) and means (b);
  • D means for displaying the molecular weight information associated with the means (c) and the antibody binding information; It is an apparatus provided with.
  • the present invention when the binding between a biological substance and an antibody is detected in association with the molecular weight, it can be performed more simply, quickly, and at a lower cost than the conventional method.
  • the present invention it is possible to detect biological substances such as proteins more easily, quickly and inexpensively than the conventional Western blotting method. That is, in detecting the target substance, it is possible to automate complicated processes such as separation of the substance and transfer to a thin film for reaction with the antibody. In addition, since it is not necessary to perform fluorescence or enzyme labeling of the antibody used for detecting the target substance, the cost for detection can be reduced.
  • the analysis algorithm of a virtual western blotting system is shown.
  • the measurement algorithm and device configuration of the virtual western blotting system are shown.
  • Measurement example with Virtual Western Blotting System Band display example of target substance by virtual western blotting system.
  • One embodiment of the present invention is an identification processing method for a substance that binds to an arbitrary antibody, (A) acquiring molecular weight information of the target substance and molecular weight information acquisition time; (B) acquiring binding information between the substance and the antibody, and antibody binding information acquisition time; (C) associating molecular weight information of the substance with antibody binding information from the information obtained in steps (a) and (b); (D) displaying the molecular weight information and antibody binding information associated in step (c); Is an identification processing method.
  • a substance that binds to an arbitrary antibody is identified in association with its molecular weight, and steps (a) to (d) can be automatically performed in a series of steps. desirable.
  • Substances to which this method is applied are not particularly limited as long as separation methods based on molecular weights have been established and have reactivity with antibodies.
  • they can be applied to biological substances such as proteins, lipids, and sugars. Application is effective, and proteins are particularly preferred.
  • the target substance to which the method of the present invention is applied may be in a single state not containing other impurities, or may be in a state mixed with many other substances.
  • the molecular weight information of the target substance is a numerical value corresponding to the molecular weight determined by gel filtration or electrophoresis, and may be the molecular weight in either a denatured or non-denatured state.
  • sociating molecular weight information and antibody binding information is associating molecular weight information of a target substance with antibody binding information of the substance, and the result may be displayed by an appropriate method such as a graph or a table. Alternatively, it may be stored as an appropriate function sequence.
  • the method for obtaining molecular weight information is not particularly limited, and examples include gel filtration column chromatography, electrophoresis (capillary electrophoresis, two-dimensional electrophoresis, isoelectric focusing), and the like.
  • a gel filtration column chromatography method is preferred.
  • molecular weight information can be obtained from the peak position of the target substance that has passed through the column.
  • molecular weight information can be obtained from the time-series peak position of the absorbance of the target substance (for example, absorbance at a wavelength of 280 nm or 220 nm).
  • a trace amount of a target substance that cannot directly detect UV absorption may be a protein standard substance with a known molecular weight that can detect UV absorption, or a fluorescently labeled standard substance and a target substance may be mixed. That is, the elution position of the target substance in the chromatogram data prepared by the gel filtration method depends on the particle diameter, column length, and flow rate used for the gel filtration, and UV absorption or fluorescence of a standard substance with a known molecular weight in advance.
  • the molecular weight information of the target substance can be converted from the relationship between the peak position on the time series of the intensity and the molecular weight.
  • molecular weight information can be obtained from the position of the substance in the electrophoresis pattern such as the mobility of the target substance separated by the molecular weight. Electrophoresis can be performed in a denatured or non-denatured state, and the molecular weight can be easily determined even for unknown substances by determining the relationship between the migration pattern and molecular weight in advance using a standard substance with a known molecular weight. Information can be acquired.
  • the binding information between the target substance and the antibody is the presence or absence of binding between the target substance and any antibody, and the binding amount when binding is confirmed.
  • Acquisition of binding information can include a method using a surface plasmon resonance method, a method using a crystal resonator, an electrochemical measurement method, and the like.
  • a crystal resonator when a crystal resonator is used, when a target substance is brought into contact with a crystal resonator to which an antibody is fixed and the target substance is bound to the antibody, a mass change occurs on the crystal resonator, and the frequency is reduced. If this decrease in frequency is detected and the degree of decrease is quantified, the amount of the target substance bound to the antibody can be calculated.
  • Patent Documents 7 and 8 Since there are many known techniques (for example, Patent Documents 7 and 8) for detecting an antigen-antibody reaction using surface plasmon resonance, those skilled in the art can easily use this technique. In some cases, a commercially available surface plasmon resonance device can also be used.
  • the electrochemical measurement utilizes the detection antibody (protein) adsorbed to the microelectrode and the measurement of the impedance change accompanying the binding of the target substance. Based on this change, the presence or absence and the abundance of the target substance are measured (for example, Patent Documents 13 and 14).
  • an area on the oscillator or chip to which no antibody is bound is appropriately selected. It is desirable to block in advance with a substance. Substances that can be used for this block include albumin derived from various animals, skim milk, ovalbumin, and the like that are used for blocking by Western blotting or the like.
  • Step (b) can be performed for each of a plurality of different antibodies, and a plurality of steps (b) may be performed in one processing step after the processing of step (a).
  • the molecular weight information acquisition time in the step (a) and the binding information acquisition time with the antibody in the step (b) are the time when the molecular weight information and the binding information are acquired, respectively. It can be specified as the elapsed time since the start.
  • the peak of the target substance is detected as a UV value.
  • the time from the start of processing to the detection of the UV value can be adopted as the molecular weight information acquisition time.
  • a crystal resonator device (QCM device) is used to acquire information on binding to an antibody
  • the resonator after the target substance is bound to the antibody on the resonator.
  • Frequency change (denoted as QCM value) may be detected as binding information with the antibody.
  • a change in SPR value may be detected as binding information
  • an electrochemical measurement device a change in impedance may be detected as binding information.
  • the time from the start of the process of this method to the detection of the change amount of the QCM value, SPR value, or impedance can be adopted as the binding information acquisition time with the antibody.
  • the binding information acquisition time between the antibody and the antibody is measured by an appropriate data processing device (for example, a personal computer), respectively, by UV value-time function, QCM value-time function sequence (or SPR value-time function examples) It may be saved as (a few examples of impedance change amount-time function) (for example, as a file in CSV format). From the stored UV value-time function sequence, a molecular weight-time function sequence is determined based on known molecular weight data and the like.
  • the QCM value-molecular weight function (or , SPR value ⁇ molecular weight function or impedance variation ⁇ molecular weight function) can be obtained and displayed by an appropriate display method (see, for example, FIG. 4).
  • the deviation between the molecular weight information acquisition time (molecular weight-time function) and the antibody binding information acquisition time (QCM value-time function, SPR value-time function or impedance change amount-time function) is corrected by an appropriate method. Also good.
  • the method for obtaining the QCM value-molecular weight function can be easily performed by those skilled in the art using an analysis algorithm as shown in FIG.
  • Another embodiment of the present invention is an apparatus used for separating a substance in a sample based on molecular weight, and detecting the molecular weight information of the substance and binding information with an arbitrary antibody in association with each other.
  • A means for obtaining molecular weight information of each separated substance and time from obtaining a sample to obtaining each molecular weight information;
  • B a means for acquiring binding information of each separated substance with an antibody and a time from sample introduction to acquisition of each antibody binding information;
  • C means for associating molecular weight information of each substance and antibody binding information from the information obtained by means (a) and means (b);
  • D means for displaying the molecular weight information associated with the means (c) and the antibody binding information; It is an apparatus provided with.
  • This embodiment is an apparatus for carrying out the above-described process identification method of the present invention.
  • the presence / absence of binding to an arbitrary antibody, the binding amount, etc. from a sample containing a large amount of contaminants Is associated with the molecular weight of the target substance and is presented as binding information.
  • a detector that detects the absorbance of each separated substance as a UV value, etc. can be used.
  • the UV value and the like are acquired together with the acquisition time, and these data are sent to a data processing apparatus (for example, a personal computer).
  • the UV value of the substance separated using appropriate software-time It may be saved as a function sequence. Further, based on the UV value-time function obtained from a substance having a known molecular weight, the molecular weight-time function of the separated substance can be obtained using appropriate software.
  • the binding information between each substance in the sample and the antibody is obtained by using the above-mentioned crystal resonator device, surface plasmon resonance device, or electrochemical measurement device as an acquisition means, and the QCM value, SPR value, or impedance change, respectively. It can be obtained as a quantity.
  • the QCM value, SPR value, or impedance change amount is acquired together with the acquisition time, and these data are sent to a data processing device (for example, a personal computer).
  • QCM value-time function sequence, SPR value-time function sequence, or impedance change amount -It may be stored as a time function sequence.
  • the stored QCM value-time function sequence, SPR value-time function sequence or impedance change amount-time function sequence is determined by the above-mentioned molecular weight (for each separated substance) -time by a data processor and appropriate software. Convert the function into a QCM value-molecular weight function, SPR value-time function or impedance change-time function, and present the result in an appropriate display format (for example, a display attached to a personal computer). Can do.
  • the apparatus of the present invention may be provided with an introduction part for introducing a sample, and may be provided with a lever or the like that can open and close the introduction port appropriately when introducing the sample.
  • the lever may be used to send the opening / closing of the introduction port as a signal to the data processing device, and when the signal is detected, the time for obtaining the molecular weight information or the binding information with the antibody may be specified.
  • any antibody that binds to the target substance is provided with an introduction port in a means for obtaining antibody binding information, such as a QCM device, a surface plasmon resonance device, or an electrochemical measurement device.
  • a target substance detection site for example, a crystal resonator of a QCM apparatus, a surface plasmon resonance apparatus, or a sensor chip of an electrochemical measurement apparatus.
  • an antibody may be introduced from a sample introduction port and attached to a detection site of a QCM device, a surface plasmon resonance device, or an electrochemical measurement device.
  • FIG. 2 shows the elements of an automated virtual western blotting system.
  • Bio-Logic Duo flow system manufactured by Bio-Rad
  • BioSelect SEC250-5 manufactured by Bio-Rad
  • a quartz crystal biosensing device QCM934 manufactured by Seiko EG & G Co.
  • a quartz resonator used a 5 mm diameter electrode with an electrode area of 0.196 cm 2 coated with gold.
  • a flow cell QCM934-510 was used for the reaction between the target substance and the antibody on the crystal resonator.
  • a phosphate buffer As a solution for adsorption cleaning, a phosphate buffer was used, and the solution was fed at a flow rate of 0.3 ml / min.
  • the antibody for detection was adsorbed on the electrode at a volume of 0.5 mg at 0.3 mg / ml.
  • the bovine serum albumin solution was adsorbed to the electrode at a volume of 0.5 ml at 3 mg / ml, and then thoroughly washed with a phosphate buffer.
  • a final concentration of 325 ⁇ g / ml of a target substance (IgG having a molecule of about 150 kDa) and a standard substance (Thyoglobulin, IgG, Ovalbumin, Myoglobin, Vitamin B12) for determining the molecular weight were mixed to a final volume of 200 ⁇ l.
  • a sample containing the target substance was applied to the apparatus shown in FIG. 2 at a flow rate of 0.9 ml / min.
  • the molecular weight can be accurately estimated as the elution time based on the absorbance of ultraviolet light (280 nm) of the mixed standard substance (Thyoglobulin, IgG, Ovalbumin, Myoglobin, Vitamin B12: Bio-Rad Gel filtration).
  • the elution time corresponding to the reaction sample (anti-IgG antibody) was 7.69 minutes (corresponding to IgG in the standard substance).
  • the upper part of FIG. 3 shows the time-series peaks of the absorbance of the mixed standard substance in the ultraviolet ray (280 nm). A decrease in the vibration frequency ( ⁇ Hz) of the QCM was observed in accordance with that time.
  • the measurement value starts to be continuously accumulated with the measurement start lever as a trigger, and when the measurement time is finished, the time function sequence of the accumulated UV value and QCM value is stored. .
  • the process moves to the automatic analysis algorithm, and each peak time is detected (differentiated once in the case of the QCM value and then processed). From the molecular weight data of the UV peak-time function and the known molecular weight marker, the molecular weight-time Find a function.
  • the QCM peak-time function can be converted into a QCM peak-molecular weight function, and the target protein can be displayed in a band shape on the molecular weight axis.
  • the result display is similar to that of the conventional Western blotting method (FIG. 4). Further, the amount of the target substance can be displayed as the band density and can be quantified as numerical data.
  • the present invention makes it possible to easily, quickly and inexpensively detect a biological substance typified by a protein or the like even in a trace amount.
  • a biological substance typified by a protein or the like even in a trace amount.
  • allergen eg ovalbumin
  • the causative protein of mad cow disease can be identified from meat.
  • disease-related proteins, pathogenic bacteria, or virus-derived proteins can be identified from body fluids such as saliva, blood, and urine. It can be used for identification of disease-related proteins and changes in expression in cultured cells and experimental animal tissues. It is expected to make great contributions not only in the field of medical biology, but also in fields such as medicine and food inspection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Acoustics & Sound (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Provided is a method for identifying a substance capable of binding to an arbitrary antibody, whereby, with respect to the reactivity with the antibody, the substance is detected in connection with the molecular weight thereof. Also provided is a device for carrying out said method. The method for identifying a substance capable of binding to an arbitrary antibody comprises: (a) a step for obtaining the molecular weight data of the target substance and time for obtaining the molecular weight data; (b) a step for obtaining the binding data of said substance and the antibody and time for obtaining the antibody binding data; (c) a step for making the connection between the molecular weight data of the substance and the antibody binding data thereof based on the data obtained in steps (a) and (b); and (d) a step for displaying the molecular weight data and the antibody binding data having been connected with each other in step (c). The device is used for carrying out said method.

Description

バーチャルウエスタンブロッティングシステムVirtual western blotting system
　本発明は、物質の微量分析方法及び微量分析装置に関する。より具体的には、本発明は、生体物質、特にタンパク質の微量分析方法及び微量分析装置に関する。 The present invention relates to a microanalysis method and a microanalysis apparatus for a substance. More specifically, the present invention relates to a microanalysis method and microanalysis apparatus for biological materials, particularly proteins.
　タンパク質、脂質、あるいは、糖などの生体物質の中には、極微量でも必須の役割を担っている物質が少なからず存在しており、これらの微量物質の生体内における動態が生体に影響を与えることがある。特に、タンパク質は、様々な機能を営んでいることが知られている。例えば、従来、タンパク質と言えば三大栄養素のひとつとして生体組織材料の補給源という認識が強かったが、近年では分解物のペプチドやアミノ酸を含め、血圧降下作用、内分泌、免疫および神経系への作用、抗酸化作用など生理活性機能面の重要性が増している。他方、タンパク質やその分解物は癌、高血圧、痴呆症などの原因のひとつに挙げられ、さらにアレルギーの原因物質にもなることから、健康・医療の分野においても注目されている。これらのタンパク質の多くは微量で作用するため、研究開発や臨床検査における検体に含まれる量もごくわずかとなることが多い。そのため、これらの分析には高度な専門技術と特別の装置が必要となる。今後、微量タンパク質成分に関する研究や検査の重要度は高まる一方であり、微量タンパク質を精度よく簡便に検出できる分析技術や自動化装置が必要とされている。 There are many substances that play an essential role even in trace amounts in biological materials such as proteins, lipids, or sugars, and the dynamics of these trace materials in the body affect the living body. Sometimes. In particular, proteins are known to have various functions. For example, in the past, protein has been recognized as a source of biological tissue material as one of the three major nutrients, but in recent years, including peptides and amino acids of degradation products, blood pressure lowering action, endocrine function, immunity, and nervous system The importance of physiologically active functions such as action and antioxidant action is increasing. On the other hand, proteins and their degradation products are listed as one of the causes of cancer, hypertension, dementia and the like, and are also a causative agent of allergies, and thus are attracting attention in the fields of health and medicine. Since many of these proteins act in trace amounts, the amount contained in specimens in research and development and clinical tests is often negligible. Therefore, these analyzes require high expertise and special equipment. In the future, the importance of research and testing regarding trace protein components will continue to increase, and there is a need for analytical techniques and automated equipment that can detect trace proteins accurately and simply.
　バイオテクノロジー分野において、ゲノム、タンパク質、細胞それぞれの機能やその量的変化を測定する基本原理はほぼ確立されている。２１世紀に入りゲノム関連の測定機器分野(例えば、ＤＮＡシークエンサー、Ｒｅａｌ－Ｔｉｍｅ　ＰＣＲ、ＤＮＡマイクロアレイなど）では大量サンプルの高速自動測定が可能で、かつ比較的安価な機器が市場に登場している。これらの機器は、研究の生産性効率化を促すとともに、これまでにはないバイオ技術を用いた医療、産業分野での応用を可能にしている。一方、タンパク質関連の汎用測定機器分野では、大量サンプルの高速自動測定を可能にする機器はまだ少ないのが現状である。
　現在行われているタンパク質の微量分析技術のひとつにウエスタンブロッティング法がある。この方法は電気泳動法によって分離したタンパク質を、検出感度の高い抗原抗体反応を利用して検出するものである。この方法は単に検出するだけでなく、タンパク質の結合状態や修飾状態なども判別できるため、生命科学分野で特に重宝されている。しかし、ウエスタンブロッティング法にも、操作の煩雑さ、測定時間、定量データの互換性などいくつか課題が残されている。ウエスタンブロッティング法では、タンパク質の分離に電気泳動法を用い、分離タンパク質の検出に免疫染色法を用いている。電気泳動法はタンパク質の分解能に優れているが、免疫染色ではタンパク質である抗体を使用するため、ゲル内で抗原と目的タンパク質とを反応させて発色させるのが難しく、一度薄膜上に転写する必要がある。このように、従来のウエスタンブロティング法においては、タンパク質を抗体で検出する場合、最終的な検出結果を確認するまでに、半日から１日程度の時間と生化学的実験に関する相当の経験と知識が必要となる。さらに従来法では、定量性や再現性に課題があり、ウエスタンブロティング法と同等の情報を有する迅速かつ簡易な技術が求められている。 In the field of biotechnology, basic principles for measuring the functions and quantitative changes of genomes, proteins, and cells are almost established. In the 21st century, in the field of genome-related measuring instruments (for example, DNA sequencers, Real-Time PCR, DNA microarrays, etc.), instruments capable of high-speed automatic measurement of a large amount of samples and relatively inexpensive have appeared on the market. These devices not only promote research productivity efficiency, but also enable applications in the medical and industrial fields using unprecedented biotechnology. On the other hand, in the field of protein-related general-purpose measuring instruments, there are still few instruments that enable high-speed automatic measurement of large samples.
One of the protein microanalysis techniques currently used is Western blotting. In this method, proteins separated by electrophoresis are detected using an antigen-antibody reaction with high detection sensitivity. This method is particularly useful in the field of life science because it can not only detect but also determine the binding state and modification state of proteins. However, the Western blotting method still has some problems such as complicated operation, measurement time, and compatibility of quantitative data. In Western blotting, electrophoresis is used for protein separation, and immunostaining is used for detection of separated protein. Electrophoresis is superior in protein resolution, but because immunostaining uses antibodies that are proteins, it is difficult to react with the antigen and the target protein in the gel to develop color, and it is necessary to transfer it to a thin film once. There is. As described above, in the conventional Western blotting method, when a protein is detected with an antibody, it takes about half a day to a day and considerable experience and knowledge about biochemical experiments until the final detection result is confirmed. Is required. Furthermore, the conventional method has problems in quantitativeness and reproducibility, and a quick and simple technique having information equivalent to that of the Western blotting method is required.
　試料中に微量に存在する物質を抗体によりできるだけ迅速かつ簡易に検出する方法として、水晶振動子を使用する方法（例えば、特許文献１～特許文献６）、表面プラズモン共鳴を使用する方法（例えば、特許文献７、特許文献８）、電気化学的測定手法（例えば、特許文献１３、１４）などが知られている。水晶振動子は、水晶の圧電効果により高周波数精度の発振を起こす受動素子である。水晶振動子を用いた物質の測定方法は、振動子表面へ物質が吸着すると、その重量に比例して周波数が変化するため、この周波数変化を利用して微量成分を検出するものである。水晶振動子表面に固定された抗体が抗原と反応すると、振動子の周波数変化によって、抗原抗体反応感知することができるため、水晶振動子を使用した検出方法は、使用する抗体を予め標識する必要がなく、従来の免疫染色的な方法に比較して簡易、迅速、かつ安価に行うことができる。また、水晶振動子を高速液体クロマトグラフィーやガスクロマトグラフィーなどと組み合わせて、物質の測定又は検出を短時間で行う方法も報告されており（例えば、特許文献９～特許文献１２）、水晶振動子を利用した検出方法や装置の開発は急速な進展を遂げている。
　しかしながら、生体物質、特にタンパク質を抗体で検出する方法の中でも、生体物質の分子量と関連づけて抗体との反応性を検出する方法、例えば、ウエスタンブロッティングのような方法については、以前として従来法に頼っているのが現状である。 As a method for detecting a substance present in a trace amount in a sample as quickly and simply as possible with an antibody, a method using a crystal resonator (for example, Patent Documents 1 to 6), a method using surface plasmon resonance (for example, Patent Documents 7 and 8), electrochemical measurement methods (for example, Patent Documents 13 and 14), and the like are known. The crystal resonator is a passive element that causes oscillation with high frequency accuracy by the piezoelectric effect of crystal. In the method for measuring a substance using a crystal resonator, when the substance is adsorbed on the surface of the vibrator, the frequency changes in proportion to its weight. Therefore, a trace component is detected using this frequency change. When the antibody immobilized on the surface of the quartz crystal reacts with the antigen, the antigen-antibody reaction can be detected by the change in frequency of the vibrator. Therefore, the detection method using the quartz crystal needs to label the antibody to be used in advance. Therefore, it can be carried out simply, quickly and inexpensively as compared with the conventional immunostaining method. In addition, a method of measuring or detecting a substance in a short time by combining a crystal resonator with high performance liquid chromatography or gas chromatography has been reported (for example, Patent Document 9 to Patent Document 12). The development of detection methods and devices using the sensor has made rapid progress.
However, among methods for detecting biological materials, particularly proteins, with antibodies, methods that detect reactivity with antibodies in relation to the molecular weight of biological materials, such as Western blotting, have relied on conventional methods. This is the current situation.
特開昭６３－１１８３５JP 63-11835 特開昭６４－３５２６９JP-A 64-35269 特開平３－７７０６１JP-A-3-77061 特開平６－１３８１２５JP-A-6-138125 特開平１０－２７４６５４JP-A-10-274654 ＷＯ２００３／１００３８６WO2003 / 100386 特開平１０－９０２７１JP-A-10-90271 特開２００９－２５０９６０JP2009-250960 特開２００４－２４５６１３JP2004-245613 ＵＳ２００６／０１７９９１８US2006 / 0179918 ＵＳ２００６／０１８３１６５US2006 / 0183165 ＵＳ６，７２７，１０４US 6,727,104 特開２００８－１０１９４９JP2008-101949 特開平５－２４０８２５JP-A-5-240825
　　本発明は、任意の抗体と結合する生体物質を、その分子量と関連づけて検出する、より簡易、迅速、かつ、安価な方法の開発を目標として鋭意研究を行った結果完成されたものである。
　従って、本発明は、任意の抗体と結合する物質の同定処理方法であって、抗体との反応性に関し、その分子量と関連づけて検出する方法の提供を目的とする。
　また、本発明は、任意の抗体と結合する物質の同定処理方法であって、抗体との反応性に関し、その分子量と関連づけて検出する装置の提供を目的とする。 The present invention has been completed as a result of earnest research aimed at developing a simpler, faster and cheaper method for detecting a biological substance that binds to an arbitrary antibody in association with its molecular weight.
Accordingly, an object of the present invention is to provide a method for identifying a substance that binds to an arbitrary antibody, and to detect a substance associated with the antibody in relation to its molecular weight.
Another object of the present invention is to provide an identification processing method for a substance that binds to an arbitrary antibody, which detects the reactivity with an antibody in association with its molecular weight.
　これまでに、水晶振動子や表面プラズモン共鳴を利用して抗原抗体反応を検出する方法及び装置、水晶振動子とクロマトグラフィーなどを組み合わせた物質の測定方法及び装置などが従来技術として報告されている。しかしながら、検出物質の抗体との反応性を分子量と関連づけて検出する方法及び装置については、これまでに知られてはいなかった。本発明者らは、物質の分子量を決定する手段としてゲルろ過カラムクロマトグラフィーを用い、抗原抗体反応の検出手段として、例えば、水晶振動子を使用したＱＣＭ（quartz crystal microbalance）装置を用い、さらに、分子量と抗体との反応性を関連づける手段を導入し、本発明を完成させた。従来法において、物質の抗体との反応性をその分子量と関連づけて１連の工程で検出する方法は報告されておらず、また、このような方法を実現する装置も知られていない。 So far, methods and devices for detecting antigen-antibody reaction using quartz crystal and surface plasmon resonance, methods and devices for measuring substances combining quartz crystal and chromatography, etc. have been reported as conventional technologies. . However, a method and an apparatus for detecting the reactivity of a detection substance with an antibody in relation to the molecular weight have not been known so far. The present inventors use gel filtration column chromatography as a means for determining the molecular weight of a substance, and as a means for detecting an antigen-antibody reaction, for example, using a QCM (quartz crystal microbalance) device using a crystal resonator, Means for associating molecular weight with antibody reactivity were introduced to complete the present invention. In the conventional method, a method for detecting the reactivity of a substance with an antibody in association with its molecular weight in a series of steps has not been reported, and an apparatus for realizing such a method is not known.
　すなわち、本発明は、任意の抗体と結合する物質の同定処理方法であって、
（ａ）目的物質の分子量情報と、分子量情報取得時間を取得する工程、
（ｂ）該物質と抗体との結合情報と、抗体結合情報取得時間を取得する工程、
（ｃ）工程（ａ）と工程（ｂ）で得られた情報から該物質の分子量情報と抗体結合情報とを関連付ける工程、
（ｄ）工程（ｃ）で関連付けられた分子量情報と抗体結合情報を表示する工程、
を含む同定処理方法である。
　さらに、試料中の物質を分子量に基づいて分離し、該物質の分子量情報及び任意の抗体との結合情報を関連付けて検出するために使用される装置であって、
（ａ）分離された各物質の分子量情報と、試料導入から各分子量情報を取得するまでの時間を取得する手段、
（ｂ）分離された各物質の抗体との結合情報と、試料導入から各抗体結合情報を取得するまでの時間を取得する手段、
（ｃ）手段（ａ）と手段（ｂ）により得られた情報から各物質の分子量情報と抗体結合情報とを関連付ける手段、
（ｄ）手段（ｃ）で関連付けられた分子量情報と抗体結合情報を表示する手段、
　を備える装置である。 That is, the present invention is an identification processing method for a substance that binds to an arbitrary antibody,
(A) acquiring molecular weight information of the target substance and molecular weight information acquisition time;
(B) acquiring binding information between the substance and the antibody, and antibody binding information acquisition time;
(C) associating molecular weight information of the substance with antibody binding information from the information obtained in steps (a) and (b);
(D) displaying the molecular weight information and antibody binding information associated in step (c);
Is an identification processing method.
Furthermore, it is an apparatus used for separating a substance in a sample based on the molecular weight, and detecting the molecular weight information of the substance and binding information with an arbitrary antibody in association with each other,
(A) means for obtaining molecular weight information of each separated substance and time from obtaining a sample to obtaining each molecular weight information;
(B) a means for acquiring binding information of each separated substance with an antibody and a time from sample introduction to acquisition of each antibody binding information;
(C) means for associating molecular weight information of each substance and antibody binding information from the information obtained by means (a) and means (b);
(D) means for displaying the molecular weight information associated with the means (c) and the antibody binding information;
It is an apparatus provided with.
　本発明により、生体物質と抗体との結合を、分子量と関連づけて検出する場合、従来法と比較して、より簡易、迅速、かつ、安価に行うことが可能となる。 According to the present invention, when the binding between a biological substance and an antibody is detected in association with the molecular weight, it can be performed more simply, quickly, and at a lower cost than the conventional method.
　本発明により、従来型のウエスタンブロッティング法に比べて、より簡易、迅速、かつ安価にタンパク質などの生体物質の検出が可能となる。すなわち、目的物質を検出するにあたり、物質の分離、抗体との反応のための薄膜への転写など、煩雑な工程を自動化することが可能である。また、目的物質の検出に使用する抗体の蛍光あるいは酵素標識等する必要がないため、検出にかかるコストを軽減することができる。 According to the present invention, it is possible to detect biological substances such as proteins more easily, quickly and inexpensively than the conventional Western blotting method. That is, in detecting the target substance, it is possible to automate complicated processes such as separation of the substance and transfer to a thin film for reaction with the antibody. In addition, since it is not necessary to perform fluorescence or enzyme labeling of the antibody used for detecting the target substance, the cost for detection can be reduced.
　本発明にかかる方法は、その工程を自動化することができるため、再現性が高い試験結果を得ることができる。また、自動化された機器は、臨床検査における試験プロトコールの標準化に貢献することができる。 Since the method according to the present invention can automate the process, a highly reproducible test result can be obtained. Automated instruments can also contribute to standardization of test protocols in clinical testing.
バーチャルウエスタンブロッティングシステムの解析アルゴリズムを示す。The analysis algorithm of a virtual western blotting system is shown. バーチャルウエスタンブロッティングシステムの計測アルゴリズムと装置構成を示す。The measurement algorithm and device configuration of the virtual western blotting system are shown. バーチャルウエスタンブロッティングシステムでの計測事例。Measurement example with Virtual Western Blotting System. バーチャルウエスタンブロッティングシステムによる目的物質のバンド表示例。Band display example of target substance by virtual western blotting system.
　本発明の実施形態の１つは、任意の抗体と結合する物質の同定処理方法であって、
（ａ）目的物質の分子量情報と、分子量情報取得時間を取得する工程、
（ｂ）該物質と抗体との結合情報と、抗体結合情報取得時間を取得する工程、
（ｃ）工程（ａ）と工程（ｂ）で得られた情報から該物質の分子量情報と抗体結合情報とを関連付ける工程、
（ｄ）工程（ｃ）で関連付けられた分子量情報と抗体結合情報を表示する工程、
を含む同定処理方法である。
　本実施形態に示される方法は、任意の抗体と結合する物質をその分子量と関連づけて同定処理するものであり、工程（ａ）～（ｄ）までを、一連の工程で自動的に行うことが望ましい。本方法が適用される物質は、分子量による分離方法が確立されており、抗体との反応性を有するものであれば、特に限定はしないが、例えば、タンパク質、脂質、糖などの生体物質への適用が効果的であり、特に、タンパク質が好ましい。本発明の方法を適用する目的物質は、他の夾雑物を含まない単一な状態であっても、あるいは、他の多くの物質と混合された状態であってもよい。目的物質の分子量情報とは、ゲルろ過法や電気泳動法などにより決定される分子量に相当する数値のことで、変性又は非変性いずれの状態における分子量であってもよい。
　なお、「関連づける」の意味は、特に、特別なことを意味するものではなく、一般に使用される意味として捉えてよい。例えば、「分子量情報と抗体結合情報を関連づける」とは、目的物質の分子量情報と該物質の抗体結合情報を対応付けることで、その結果は、グラフや表等、適当な方法で表示してもよく、又は、適当な関数列などとして保存しておいてもよい。 One embodiment of the present invention is an identification processing method for a substance that binds to an arbitrary antibody,
(A) acquiring molecular weight information of the target substance and molecular weight information acquisition time;
(B) acquiring binding information between the substance and the antibody, and antibody binding information acquisition time;
(C) associating molecular weight information of the substance with antibody binding information from the information obtained in steps (a) and (b);
(D) displaying the molecular weight information and antibody binding information associated in step (c);
Is an identification processing method.
In the method shown in this embodiment, a substance that binds to an arbitrary antibody is identified in association with its molecular weight, and steps (a) to (d) can be automatically performed in a series of steps. desirable. Substances to which this method is applied are not particularly limited as long as separation methods based on molecular weights have been established and have reactivity with antibodies. For example, they can be applied to biological substances such as proteins, lipids, and sugars. Application is effective, and proteins are particularly preferred. The target substance to which the method of the present invention is applied may be in a single state not containing other impurities, or may be in a state mixed with many other substances. The molecular weight information of the target substance is a numerical value corresponding to the molecular weight determined by gel filtration or electrophoresis, and may be the molecular weight in either a denatured or non-denatured state.
Note that the meaning of “associate” does not particularly mean anything special, and may be regarded as a commonly used meaning. For example, “associating molecular weight information and antibody binding information” is associating molecular weight information of a target substance with antibody binding information of the substance, and the result may be displayed by an appropriate method such as a graph or a table. Alternatively, it may be stored as an appropriate function sequence.
　分子量情報を取得する方法は、特に限定はされず、例えば、ゲルろ過カラムクロマトグラフィー法、電気泳動法（キャピラリー電気泳動、二次元電気泳動、等電点電気泳動）、などを挙げることができ、好ましくは、ゲルろ過カラムクロマトグラフィー法である。例えば、ゲルろ過カラムクロマトグラフィーを使用する場合、カラムを通過してきた目的物質のピーク位置から分子量情報を求めることができる。例えば、目的物質の吸光度（例えば２８０ｎｍあるいは２２０ｎｍの波長の吸光度）の時系列上のピーク位置から分子量情報を求めることができる。直接的に紫外線吸収が検出できない微量な目的物質は、予め分子量が既知の紫外線吸収が検出可能なタンパク質標準物質、あるいは蛍光標識した標準物質と目的物質を混合してもよい。すなわち、ゲルろ過法により作製されるクロマトグラムデータ内での目的物質の溶出位置は、ゲルろ過に使用した粒子径、カラム長、流速に依存し、予め分子量が既知の標準物質の紫外線吸収あるいは蛍光強度の時系列上のピーク位置と分子量との関係から、目的物質の分子量情報を換算することができる。また、電気泳動を使用する場合、分子量によって分離された目的物質の移動度などの電気泳動パターンにおける該物質の位置から、分子量情報を求めることができる。電気泳動は、泳動対象が変性状態でも非変性状態でもよく、あらかじめ、分子量の既知の標準物質により泳動パターンと分子量との関係を決定しておくことで、未知の物質であっても容易に分子量情報を取得することができる。 The method for obtaining molecular weight information is not particularly limited, and examples include gel filtration column chromatography, electrophoresis (capillary electrophoresis, two-dimensional electrophoresis, isoelectric focusing), and the like. A gel filtration column chromatography method is preferred. For example, when gel filtration column chromatography is used, molecular weight information can be obtained from the peak position of the target substance that has passed through the column. For example, molecular weight information can be obtained from the time-series peak position of the absorbance of the target substance (for example, absorbance at a wavelength of 280 nm or 220 nm). A trace amount of a target substance that cannot directly detect UV absorption may be a protein standard substance with a known molecular weight that can detect UV absorption, or a fluorescently labeled standard substance and a target substance may be mixed. That is, the elution position of the target substance in the chromatogram data prepared by the gel filtration method depends on the particle diameter, column length, and flow rate used for the gel filtration, and UV absorption or fluorescence of a standard substance with a known molecular weight in advance. The molecular weight information of the target substance can be converted from the relationship between the peak position on the time series of the intensity and the molecular weight. When electrophoresis is used, molecular weight information can be obtained from the position of the substance in the electrophoresis pattern such as the mobility of the target substance separated by the molecular weight. Electrophoresis can be performed in a denatured or non-denatured state, and the molecular weight can be easily determined even for unknown substances by determining the relationship between the migration pattern and molecular weight in advance using a standard substance with a known molecular weight. Information can be acquired.
　目的物質と抗体との結合情報とは、目的物質と任意の抗体との結合の有無、結合が確認される場合には、結合量のことである。結合情報の取得は、表面プラズモン共鳴法を使用する方法、水晶振動子を使用する方法、電気化学的測定手法などを挙げることができる。例えば、水晶振動子を使用する場合、抗体を固定した水晶振動子に、目的物質を接触させ、目的物質が抗体と結合すると、水晶振動子上において質量変化が生じ、周波数が低下する。この周波数の低下を検出し、低下の程度を数値化すれば、抗体と結合した目的物質の量を算定することが可能となる。このような水晶振動子を利用した抗原抗体反応の検出技術は、公知の技術としても多く報告されており（例えば、特許文献１～６などを参照のこと）、当業者であれば容易に利用することができる。また、場合によっては、市販の水晶振動子装置（ＱＣＭ装置）を使用することもできる。
　表面プラズモン共鳴を使用した方法は、任意の抗体を固定したセンサーチップと目的物質を接触させ、抗体に目的物質が結合すると表面プラズモン共鳴（ＳＰＲ）が変化することを利用したものである。この変化に基づいて、目的物質の有無や存在量を測定する。表面プラズモン共鳴を使用した抗原抗体反応の検出技術も、多くの公知技術（例えば、特許文献７、８など）が存在するため、当業者であれば当該技術を容易に使用することができる。また、場合によっては、市販の表面プラズモン共鳴装置を使用することもできる。
　電気化学的測定は検出抗体（タンパク質）を微小電極に吸着させ、目的物質の結合に伴うインピーダンス変化を測定することを利用したものである。この変化に基づいて、目的物質の有無や存在量を測定する（例えば、特許文献１３、１４）。
　水晶振動子、表面プラズモン共鳴又は電気化学的手法のセンサーチップに対し、抗体を介さない非特異的な吸着が生じることを防ぐために、抗体が結合してない振動子又はチップ上の領域を適当な物質で予めブロックしておくことが望ましい。このブロックに使用可能な物質は、ウエスタンブロッティング法などでブロッキングに使用される、例えば、各種動物由来のアルブミン、スキムミルク、オブアルブミン、などが利用可能である。 The binding information between the target substance and the antibody is the presence or absence of binding between the target substance and any antibody, and the binding amount when binding is confirmed. Acquisition of binding information can include a method using a surface plasmon resonance method, a method using a crystal resonator, an electrochemical measurement method, and the like. For example, when a crystal resonator is used, when a target substance is brought into contact with a crystal resonator to which an antibody is fixed and the target substance is bound to the antibody, a mass change occurs on the crystal resonator, and the frequency is reduced. If this decrease in frequency is detected and the degree of decrease is quantified, the amount of the target substance bound to the antibody can be calculated. Many detection techniques for antigen-antibody reaction using such a quartz oscillator have been reported as known techniques (see, for example, Patent Documents 1 to 6), and those skilled in the art can easily use them. can do. In some cases, a commercially available crystal resonator device (QCM device) can also be used.
The method using surface plasmon resonance utilizes the fact that the surface plasmon resonance (SPR) changes when a target substance is brought into contact with a sensor chip in which an arbitrary antibody is immobilized and the target substance is bound. Based on this change, the presence / absence and abundance of the target substance are measured. Since there are many known techniques (for example, Patent Documents 7 and 8) for detecting an antigen-antibody reaction using surface plasmon resonance, those skilled in the art can easily use this technique. In some cases, a commercially available surface plasmon resonance device can also be used.
The electrochemical measurement utilizes the detection antibody (protein) adsorbed to the microelectrode and the measurement of the impedance change accompanying the binding of the target substance. Based on this change, the presence or absence and the abundance of the target substance are measured (for example, Patent Documents 13 and 14).
In order to prevent non-specific adsorption that does not involve antibodies to a crystal oscillator, surface plasmon resonance, or electrochemical sensor chip, an area on the oscillator or chip to which no antibody is bound is appropriately selected. It is desirable to block in advance with a substance. Substances that can be used for this block include albumin derived from various animals, skim milk, ovalbumin, and the like that are used for blocking by Western blotting or the like.
　工程（ｂ）は、異なる複数の抗体ごとに行うことができ、工程（ａ）の処理後に、複数の工程（ｂ）を１つの処理工程において行ってもよい。工程（ａ）の分子量情報取得時間、工程（ｂ）の抗体との結合情報取得時間とは、各々、上記分子量情報、上記結合情報を取得した時間のことで、例えば、本発明の処理方法を開始してからの経過時間として特定することができる。本発明の処理方法において、例えば、分子量情報の取得のためにゲルろ過カラムクロマトグラフィーを行い、目的物質がゲルろ過カラムを通過した後、目的物質のピークをＵＶ値として検出する場合、本方法の処理開始からＵＶ値の検出までの時間を分子量情報取得時間として採用することができる。また、本発明の処理方法において、抗体との結合情報の取得のために、例えば、水晶振動子装置（ＱＣＭ装置）を使用する場合、目的物質が振動子上の抗体と結合した後の振動子の周波数変化（ＱＣＭ値と記す）を抗体との結合情報として検出してもよい。また、表面プラズモン共鳴装置を使用する場合には、ＳＰＲ値の変化を結合情報とし、電気化学的測定装置を使用する場合には、インピーダンス変化を結合情報として検出してもよい。この場合、本方法の処理開始からＱＣＭ値、ＳＰＲ値又はインピーダンスの変化量の検出までの時間を、抗体との結合情報取得時間として採用することができる。 Step (b) can be performed for each of a plurality of different antibodies, and a plurality of steps (b) may be performed in one processing step after the processing of step (a). The molecular weight information acquisition time in the step (a) and the binding information acquisition time with the antibody in the step (b) are the time when the molecular weight information and the binding information are acquired, respectively. It can be specified as the elapsed time since the start. In the treatment method of the present invention, for example, when gel filtration column chromatography is performed to obtain molecular weight information, and the target substance passes through the gel filtration column, the peak of the target substance is detected as a UV value. The time from the start of processing to the detection of the UV value can be adopted as the molecular weight information acquisition time. In the processing method of the present invention, for example, when a crystal resonator device (QCM device) is used to acquire information on binding to an antibody, the resonator after the target substance is bound to the antibody on the resonator. Frequency change (denoted as QCM value) may be detected as binding information with the antibody. Further, when using a surface plasmon resonance device, a change in SPR value may be detected as binding information, and when using an electrochemical measurement device, a change in impedance may be detected as binding information. In this case, the time from the start of the process of this method to the detection of the change amount of the QCM value, SPR value, or impedance can be adopted as the binding information acquisition time with the antibody.
　本発明の方法により検出された目的物質の分子量情報を換算するための、例えば、ＵＶ値と分子量取得時間、並びに、任意の抗体と目的物質との結合情報を換算するための、例えば、ＱＣＭ値と抗体との結合情報取得時間は、適当なデータ処理装置（例えば、パーソナルコンピュータ）に、各々、ＵＶ値－時間関数、ＱＣＭ値－時間関数の数列（あるいは、ＳＰＲ値－時間関数の数例もしくはインピーダンス変化量－時間関数の数例）（例えば、ＣＳＶ形式などのファイルとして）として保存してもよい。保存されたＵＶ値－時間関数列から、既知の分子量データ等に基づいて、分子量－時間関数列を求める。このようにして求められた、分子量－時間関数列とＱＣＭ値－時間関数列（あるいは、ＳＰＲ値－時間関数例もしくはインピーダンス変化量－時間関数例）を利用して、ＱＣＭ値－分子量関数（あるいは、ＳＰＲ値－分子量関数もしくはインピーダンス変化量－分子量関数）を求め、適当な表示方法（例えば、図４を参照）で表示することができる。このとき、分子量情報取得時間（分子量－時間関数）と抗体結合情報取得時間（ＱＣＭ値－時間関数、ＳＰＲ値－時間関数又はインピーダンス変化量－時間関数）のズレを適当な方法により補正を行ってもよい。ＱＣＭ値－分子量関数を求める方法は、図１に示すような解析アルゴリズムを用いて、当業者であれば容易に実施することが可能である。 For converting the molecular weight information of the target substance detected by the method of the present invention, for example, for converting the UV value and the molecular weight acquisition time, and the binding information between any antibody and the target substance, for example, the QCM value The binding information acquisition time between the antibody and the antibody is measured by an appropriate data processing device (for example, a personal computer), respectively, by UV value-time function, QCM value-time function sequence (or SPR value-time function examples) It may be saved as (a few examples of impedance change amount-time function) (for example, as a file in CSV format). From the stored UV value-time function sequence, a molecular weight-time function sequence is determined based on known molecular weight data and the like. Using the molecular weight-time function sequence and the QCM value-time function sequence (or SPR value-time function example or impedance change amount-time function example) thus determined, the QCM value-molecular weight function (or , SPR value−molecular weight function or impedance variation−molecular weight function) can be obtained and displayed by an appropriate display method (see, for example, FIG. 4). At this time, the deviation between the molecular weight information acquisition time (molecular weight-time function) and the antibody binding information acquisition time (QCM value-time function, SPR value-time function or impedance change amount-time function) is corrected by an appropriate method. Also good. The method for obtaining the QCM value-molecular weight function can be easily performed by those skilled in the art using an analysis algorithm as shown in FIG.
　本発明の他の実施形態は、試料中の物質を分子量に基づいて分離し、該物質の分子量情報及び任意の抗体との結合情報を関連付けて検出するために使用される装置であって、
（ａ）分離された各物質の分子量情報と、試料導入から各分子量情報を取得するまでの時間を取得する手段、
（ｂ）分離された各物質の抗体との結合情報と、試料導入から各抗体結合情報を取得するまでの時間を取得する手段、
（ｃ）手段（ａ）と手段（ｂ）により得られた情報から各物質の分子量情報と抗体結合情報とを関連付ける手段、
（ｄ）手段（ｃ）で関連付けられた分子量情報と抗体結合情報を表示する手段、
　を備える装置である。
　本実施形態は、上述の本発明の処理同定方法を実施するための装置であり、目的物質の他、共雑物質を多く含む試料の中から、任意の抗体との結合の有無、結合量などを結合情報として、目的物質の分子量と関連付けて提示する装置である。試料の分離手段及び分離された各物質の分子量情報の取得手段として、上述のゲルろ過カラムクロマトグラフィー、電気泳動装置を、分離された各物質の吸光度等をＵＶ値などとして検出する検出器を組み合わせて使用することができる。ＵＶ値等は、その取得時間と共に取得し、これらのデータをデータ処理装置（例えば、パーソナルコンピュータ）へ送り、データ処理装置において、適当なソフトトウェアを使用して分離された物質のＵＶ値－時間関数列として保存してもよい。さらに、分子量既知の物質から求められるＵＶ値－時間関数に基づいて、分離された物質の分子量－時間関数を適当なソフトウェアを使用して求めることができる。
　また、試料中の各物質と抗体との結合情報は、上述の水晶振動子装置、表面プラズモン共鳴装置又は電気化学的測定装置などを取得手段として使用し、各々、ＱＣＭ値、ＳＰＲ値又はインピーダンス変化量などとして取得することができる。ＱＣＭ値、ＳＰＲ値又はインピーダンス変化量は、その取得時間と共に取得し、これらのデータをデータ処理装置（例えば、パーソナルコンピュータ）へ送りＱＣＭ値－時間関数列、ＳＰＲ値－時間関数列又はインピーダンス変化量－時間関数列として保存してもよい。ここで、保存されるＱＣＭ値－時間関数列、ＳＰＲ値－時間関数列又はインピーダンス変化量－時間関数列は、データ処理装置と適当なソフトウェアにより、上記分子量（分離された各物質の）－時間関数から、ＱＣＭ値－分子量関数、ＳＰＲ値－時間関数又はインピーダンス変化量－時間関数に換算し、その結果を適当な表示形式で提示部（例えば、パーソナルコンピュータに付属したディスプレイなど）において提示することができる。 Another embodiment of the present invention is an apparatus used for separating a substance in a sample based on molecular weight, and detecting the molecular weight information of the substance and binding information with an arbitrary antibody in association with each other.
(A) means for obtaining molecular weight information of each separated substance and time from obtaining a sample to obtaining each molecular weight information;
(B) a means for acquiring binding information of each separated substance with an antibody and a time from sample introduction to acquisition of each antibody binding information;
(C) means for associating molecular weight information of each substance and antibody binding information from the information obtained by means (a) and means (b);
(D) means for displaying the molecular weight information associated with the means (c) and the antibody binding information;
It is an apparatus provided with.
This embodiment is an apparatus for carrying out the above-described process identification method of the present invention. In addition to the target substance, the presence / absence of binding to an arbitrary antibody, the binding amount, etc., from a sample containing a large amount of contaminants Is associated with the molecular weight of the target substance and is presented as binding information. Combined with the above-mentioned gel filtration column chromatography and electrophoresis device as a means for separating the sample and the molecular weight information of each separated substance, combined with a detector that detects the absorbance of each separated substance as a UV value, etc. Can be used. The UV value and the like are acquired together with the acquisition time, and these data are sent to a data processing apparatus (for example, a personal computer). In the data processing apparatus, the UV value of the substance separated using appropriate software-time It may be saved as a function sequence. Further, based on the UV value-time function obtained from a substance having a known molecular weight, the molecular weight-time function of the separated substance can be obtained using appropriate software.
In addition, the binding information between each substance in the sample and the antibody is obtained by using the above-mentioned crystal resonator device, surface plasmon resonance device, or electrochemical measurement device as an acquisition means, and the QCM value, SPR value, or impedance change, respectively. It can be obtained as a quantity. The QCM value, SPR value, or impedance change amount is acquired together with the acquisition time, and these data are sent to a data processing device (for example, a personal computer). QCM value-time function sequence, SPR value-time function sequence, or impedance change amount -It may be stored as a time function sequence. Here, the stored QCM value-time function sequence, SPR value-time function sequence or impedance change amount-time function sequence is determined by the above-mentioned molecular weight (for each separated substance) -time by a data processor and appropriate software. Convert the function into a QCM value-molecular weight function, SPR value-time function or impedance change-time function, and present the result in an appropriate display format (for example, a display attached to a personal computer). Can do.
　さらに本発明の装置には、試料を導入する導入部を具備しており、試料の導入を行うにあたり、導入口を適宜開閉できるようなレバーなどを備えていてもよく。該レバーにより導入口の開閉を信号としてデータ処理装置に送り、該信号の検出時を試料導入時として、分子量情報や抗体との結合情報の取得時間を特定してもよい。
　また、目的物質と結合する任意の抗体は、抗体結合情報を取得するための手段、例えば、ＱＣＭ装置、表面プラズモン共鳴装置又は電気化学的測定装置などに導入口を設け、かかる導入口から、抗体を該装置に導入し、目的物質の検出部位（例えば、ＱＣＭ装置の水晶振動子、表面プラズモン共鳴装置又は電気化学的測定装置のセンサーチップなど）に付着させてもよい。あるいは、試料の導入口から抗体を導入し、ＱＣＭ装置、表面プラズモン共鳴装置又は電気化学的測定装置の検出部位に付着させてもよい。 Furthermore, the apparatus of the present invention may be provided with an introduction part for introducing a sample, and may be provided with a lever or the like that can open and close the introduction port appropriately when introducing the sample. The lever may be used to send the opening / closing of the introduction port as a signal to the data processing device, and when the signal is detected, the time for obtaining the molecular weight information or the binding information with the antibody may be specified.
In addition, any antibody that binds to the target substance is provided with an introduction port in a means for obtaining antibody binding information, such as a QCM device, a surface plasmon resonance device, or an electrochemical measurement device. May be introduced into the apparatus and attached to a target substance detection site (for example, a crystal resonator of a QCM apparatus, a surface plasmon resonance apparatus, or a sensor chip of an electrochemical measurement apparatus). Alternatively, an antibody may be introduced from a sample introduction port and attached to a detection site of a QCM device, a surface plasmon resonance device, or an electrochemical measurement device.
　次に本発明を具体例によって説明するがこれらの例によって本発明が限定されるものではない。 Next, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples.
　図２は、自動化されたバーチャルウエスタンブロッティングシステムの要素を示す。ポンプ、サンプルアプライ、紫外線（２８０ｎｍ）の吸光度計は、Ｂｉｏ－Ｌｏｇｉｃ　Ｄｕｏ　ｆｌｏｗシステム（Ｂｉｏ－Ｒａｄ社製）を用いた。ゲルろ過ゲルろ過クロマトグラフィーとしては、Ｂｉｏ　Ｓｉｌｅｃｔ　ＳＥＣ２５０－５（Ｂｉｏ－Ｒａｄ社製）を使用した。水晶振動子バイオセンシング装置ＱＣＭ９３４（セイコー・イージーアンドジー社製）をカラムークロマトグラフィィーと紫外線吸光度計の後に結合した。水晶振動子（セイコー・イージーアンドジー社製）は、金を電極分にコートした直径５ｍｍ、電極面積０．１９６ｃｍ２を使用した。目的物質と水晶振動子上の抗体との反応はフロー型セルＱＣＭ９３４－５１０を使用した。 FIG. 2 shows the elements of an automated virtual western blotting system. Bio-Logic Duo flow system (manufactured by Bio-Rad) was used as a pump, sample apply, and ultraviolet (280 nm) absorbance meter. Gel filtration As the gel filtration chromatography, BioSelect SEC250-5 (manufactured by Bio-Rad) was used. A quartz crystal biosensing device QCM934 (manufactured by Seiko EG & G Co.) was coupled after column chromatography and an ultraviolet absorbance meter. A quartz resonator (manufactured by Seiko EG & G Co.) used a 5 mm diameter electrode with an electrode area of 0.196 cm 2 coated with gold. A flow cell QCM934-510 was used for the reaction between the target substance and the antibody on the crystal resonator.
　吸着洗浄用の溶液としては、リン酸緩衝液を用い、０．３ｍｌ／分の流速で送液した。検出用抗体は０．３ｍｇ／ｍｌで０．５ｍｌの容量で電極に吸着させた。さらにウシ血清アルブミン溶液は３ｍｇ／ｍｌで０．５ｍｌの容量で電極に吸着させたのち、リン酸緩衝液で充分洗浄した。目的物質（分子約１５０ｋＤａのＩｇＧ）最終濃度３２５μｇ／ｍｌと分子量を決定する標準物質（Ｔｈｙｏｇｌｏｂｕｌｉｎ，ＩｇＧ，Ｏｖａｌｂｕｍｉｎ，　Ｍｙｏｇｌｏｂｉｎ，Ｖｉｔａｍｉｎ　Ｂ１２）を　混合して最終容積を２００μｌとした。図２に示す機器に流速０．９ｍｌ／ｍｉｎで目的物質を含むサンプルをアプライした。 As a solution for adsorption cleaning, a phosphate buffer was used, and the solution was fed at a flow rate of 0.3 ml / min. The antibody for detection was adsorbed on the electrode at a volume of 0.5 mg at 0.3 mg / ml. Further, the bovine serum albumin solution was adsorbed to the electrode at a volume of 0.5 ml at 3 mg / ml, and then thoroughly washed with a phosphate buffer. A final concentration of 325 μg / ml of a target substance (IgG having a molecule of about 150 kDa) and a standard substance (Thyoglobulin, IgG, Ovalbumin, Myoglobin, Vitamin B12) for determining the molecular weight were mixed to a final volume of 200 μl. A sample containing the target substance was applied to the apparatus shown in FIG. 2 at a flow rate of 0.9 ml / min.
　混合した標準物質（Ｔｈｙｏｇｌｏｂｕｌｉｎ，ＩｇＧ，Ｏｖａｌｂｕｍｉｎ，Ｍｙｏｇｌｏｂｉｎ，Ｖｉｔａｍｉｎ　Ｂ１２：Ｂｉｏ－Ｒａｄ　Ｇｅｌ　ｆｉｌｔｒａｔｉｏｎ）の紫外線（２８０ｎｍ）の吸光度により、分子量を正確に溶出時間として推定することができる。本系では、反応サンプル（抗ＩｇＧ抗体）に対応する溶出時間は７．６９分（標準物質中のＩｇＧに対応）であった。図３上段は混合した標準物質の紫外線（２８０ｎｍ）の吸光度の時系列上のピークを示す。その時間と一致してＱＣＭの振動周波数の低下（ΔＨｚ）が観察された。このことは、分子量１５０ＫＤａに抗体と反応する物質（目的物質）が存在することと同質の情報を得ることができることを示している。
　パーソナルコンピュータ側では、図２の計測アルゴリズムに示したように、測定開始レバーをトリガとして測定値を連続に蓄積しはじめ、測定時間が終了すると蓄積したＵＶ値とＱＣＭ値の時間関数列を保存する。測定が終了すると自動的解析アルゴリズムに移行し、それぞれのピーク時間を検出し（ＱＣＭ値の場合一度微分してから処理）、ＵＶピーク－時間関数と既知の分子量マーカの分子量データから、分子量－時間関数を求める。この関数を利用してＱＣＭピーク－時間関数をＱＣＭピーク－分子量関数に換算して、分子量軸に目的とするタンパク質をバンド状に表示することができる。従来のウエスタンブロッティング法と似た結果表示となる（図４）。また目的物質の量は、バンドの濃さとして表示することができるとともの数値データとして定量化できる。 The molecular weight can be accurately estimated as the elution time based on the absorbance of ultraviolet light (280 nm) of the mixed standard substance (Thyoglobulin, IgG, Ovalbumin, Myoglobin, Vitamin B12: Bio-Rad Gel filtration). In this system, the elution time corresponding to the reaction sample (anti-IgG antibody) was 7.69 minutes (corresponding to IgG in the standard substance). The upper part of FIG. 3 shows the time-series peaks of the absorbance of the mixed standard substance in the ultraviolet ray (280 nm). A decrease in the vibration frequency (ΔHz) of the QCM was observed in accordance with that time. This indicates that information having the same quality as that of a substance (target substance) that reacts with an antibody having a molecular weight of 150 KDa can be obtained.
On the personal computer side, as shown in the measurement algorithm of FIG. 2, the measurement value starts to be continuously accumulated with the measurement start lever as a trigger, and when the measurement time is finished, the time function sequence of the accumulated UV value and QCM value is stored. . When the measurement is completed, the process moves to the automatic analysis algorithm, and each peak time is detected (differentiated once in the case of the QCM value and then processed). From the molecular weight data of the UV peak-time function and the known molecular weight marker, the molecular weight-time Find a function. Using this function, the QCM peak-time function can be converted into a QCM peak-molecular weight function, and the target protein can be displayed in a band shape on the molecular weight axis. The result display is similar to that of the conventional Western blotting method (FIG. 4). Further, the amount of the target substance can be displayed as the band density and can be quantified as numerical data.
　本発明は、タンパク質などに代表される生体物質を微量であっても、簡易、迅速、かつ、安価に検出することを実現ならしめるものである。現在食品検査で行われているアレルゲン（例えば卵白アルブミン）の食品中での存在を誰でも確認することができる。狂牛病の原因タンパク質であるプリオンを食肉中より同定することができる。また唾液、血液、尿中などの体液中から疾患関連タンパク質や病原性細菌あるいはウイルス由来のタンパク質を同定することができる。培養細胞や実験動物組織における疾患関連タンパク質の同定や発現変動に利用することができる。医学生物学の学問研究分野のみならず、医療、食品検査などの分野において、大いに貢献することが期待される。 The present invention makes it possible to easily, quickly and inexpensively detect a biological substance typified by a protein or the like even in a trace amount. Anyone can confirm the presence of allergen (eg ovalbumin) currently used in food inspection in food. Prion, the causative protein of mad cow disease, can be identified from meat. In addition, disease-related proteins, pathogenic bacteria, or virus-derived proteins can be identified from body fluids such as saliva, blood, and urine. It can be used for identification of disease-related proteins and changes in expression in cultured cells and experimental animal tissues. It is expected to make great contributions not only in the field of medical biology, but also in fields such as medicine and food inspection.
１１　ゲル濾過カラム
１２　電気泳動用ゲル
２１　ＱＣＭ装置（水晶振動子装置）
２２　表面プラズモン共鳴装置
２３　電気化学的測定装置 11 Gel filtration column 12 Gel for electrophoresis 21 QCM device (quartz crystal device)
22 Surface plasmon resonance device 23 Electrochemical measurement device

Claims (14)

  1. 　任意の抗体と結合する物質の同定処理方法であって、
    （ａ）目的物質の分子量情報と、分子量情報取得時間を取得する工程、
    （ｂ）該物質と抗体との結合情報と、抗体結合情報取得時間を取得する工程、
    （ｃ）工程（ａ）と工程（ｂ）で得られた情報から該物質の分子量情報と抗体結合情報とを関連付ける工程、
    （ｄ）工程（ｃ）で関連付けられた分子量情報と抗体結合情報を表示する工程、
    を含む同定処理方法。     An identification processing method for a substance that binds to an arbitrary antibody,
    (A) acquiring molecular weight information of the target substance and molecular weight information acquisition time;
    (B) acquiring binding information between the substance and the antibody, and antibody binding information acquisition time;
    (C) associating molecular weight information of the substance with antibody binding information from the information obtained in steps (a) and (b);
    (D) displaying the molecular weight information and antibody binding information associated in step (c);
    An identification processing method comprising:
  2. 　前記物質の抗体結合情報が、水晶振動子を使用した方法で取得されることを特徴とする請求項１に記載の物質の同定処理方法。 2. The substance identification processing method according to claim 1, wherein the antibody binding information of the substance is acquired by a method using a crystal resonator.
  3. 　前記物質の抗体結合情報が、表面プラズモン共鳴を使用した方法で取得されることを特徴とする請求項１に記載の物質の同定処理方法。 2. The substance identification processing method according to claim 1, wherein the antibody binding information of the substance is acquired by a method using surface plasmon resonance.
  4. 　前記物質の抗体結合情報が、電気化学的測定手法を使用した方法で取得されることを特徴とする請求項１に記載の物質の同定処理方法。 2. The substance identification processing method according to claim 1, wherein the antibody binding information of the substance is acquired by a method using an electrochemical measurement technique.
  5. 　前記分子量情報が、目的物質をゲルろ過カラムクロマトグラフィーで分離し、該カラムクロマトグラフィーにより作成されたクロマトデータにおける目的物質のピーク位置に基づいて取得されることを特徴とする請求項１乃至４のいずれかに記載の同定処理方法。 The molecular weight information is obtained based on a peak position of the target substance in chromatographic data generated by the column chromatography after the target substance is separated by gel filtration column chromatography. The identification processing method according to any one of the above.
  6. 　前記分子量情報が、目的物質を電気泳動で分離し、該電気泳動パターンにおける目的物質の位置に基づいて取得されることを特徴とする請求項１乃至４のいずれかに記載の同定処理方法。 5. The identification processing method according to claim 1, wherein the molecular weight information is acquired based on a position of the target substance in the electrophoresis pattern after separating the target substance by electrophoresis.
  7. 　前記物質がタンパク質であることを特徴とする請求項１乃至６のいずれかに記載の同定処理方法。 The identification processing method according to any one of claims 1 to 6, wherein the substance is a protein.
  8. 　試料中の物質を分子量に基づいて分離し、該物質の分子量情報及び任意の抗体との結合情報を関連付けて検出するために使用される装置であって、
    （ａ）分離された各物質の分子量情報と、試料導入から各分子量情報を取得するまでの時間を取得する手段、
    （ｂ）分離された各物質の抗体との結合情報と、試料導入から各抗体結合情報を取得するまでの時間を取得する手段、
    （ｃ）手段（ａ）と手段（ｂ）により得られた情報から各物質の分子量情報と抗体結合情報とを関連付ける手段、
    （ｄ）手段（ｃ）で関連付けられた分子量情報と抗体結合情報を表示する手段、
    　を備える装置。     An apparatus used for separating a substance in a sample based on a molecular weight, and detecting the molecular weight information of the substance and binding information with an arbitrary antibody in association with each other,
    (A) means for obtaining molecular weight information of each separated substance and time from obtaining a sample to obtaining each molecular weight information;
    (B) a means for acquiring binding information of each separated substance with an antibody and a time from sample introduction to acquisition of each antibody binding information;
    (C) means for associating molecular weight information of each substance and antibody binding information from the information obtained by means (a) and means (b);
    (D) means for displaying the molecular weight information associated with the means (c) and the antibody binding information;
    A device comprising:
  9. 　前記物質の抗体結合情報の取得手段が、水晶振動子を使用した手段であることを特徴とする請求項８に記載の装置。 The apparatus according to claim 8, wherein the means for obtaining antibody binding information of the substance is a means using a crystal resonator.
  10. 　前記物質の抗体結合情報の取得手段が、表面プラズモン共鳴を使用した手段であることを特徴とする請求項８に記載の装置。 The apparatus according to claim 8, wherein the means for acquiring antibody binding information of the substance is means using surface plasmon resonance.
  11. 　前記物質の抗体結合情報の取得手段が、電気化学的測定手法を使用した手段であることを特徴とする請求項８に記載の装置。 The apparatus according to claim 8, wherein the means for acquiring antibody binding information of the substance is a means using an electrochemical measurement technique.
  12. 　前記分子量情報の取得手段が、ゲルろ過カラムクロマトグラフィーであり、カラムクロマトグラフィーにより作成されたクロマトデータにおける物質のピーク位置に基づいて分子量情報を取得することを特徴とする請求項８乃至１１のいずれかに記載の装置。 12. The molecular weight information acquisition means is gel filtration column chromatography, and the molecular weight information is acquired based on a peak position of a substance in chromatographic data created by column chromatography. A device according to the above.
  13. 　前記分子量情報の取得手段が、電気泳動であり、電気泳動パターンにおける目的物質の位置に基づいて分子量情報を取得することを特徴とする請求項８乃至１１のいずれかに記載に記載の装置。 The apparatus according to any one of claims 8 to 11, wherein the molecular weight information acquisition unit is electrophoresis, and acquires molecular weight information based on a position of a target substance in an electrophoresis pattern.
  14. 　前記試料中の物質にタンパク質が含まれることを特徴とする請求項８乃至１３のいずれかに記載の装置。 14. The apparatus according to claim 8, wherein the substance in the sample contains a protein.
PCT/JP2010/057569 2010-04-28 2010-04-28 Virtual western blotting system WO2011135692A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2010/057569 WO2011135692A1 (en) 2010-04-28 2010-04-28 Virtual western blotting system
JP2012512589A JP5685777B2 (en) 2010-04-28 2010-04-28 Virtual western blotting system

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2010/057569 WO2011135692A1 (en) 2010-04-28 2010-04-28 Virtual western blotting system

Publications (1)

Publication Number Publication Date
WO2011135692A1 true WO2011135692A1 (en) 2011-11-03

Family

ID=44861036

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/057569 WO2011135692A1 (en) 2010-04-28 2010-04-28 Virtual western blotting system

Country Status (2)

Country Link
JP (1) JP5685777B2 (en)
WO (1) WO2011135692A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11183479A (en) * 1997-10-16 1999-07-09 Fuji Electric Co Ltd Sensor for measuring solution and method for measuring solution component
JP2002122600A (en) * 2000-10-12 2002-04-26 Nippon Laser & Electronics Lab Sensor chip for biosensor
WO2005038457A1 (en) * 2003-10-15 2005-04-28 Daiichi Pure Chemicals Co., Ltd. Method of separating and assaying adiponectin multimer
JP2007518074A (en) * 2003-12-30 2007-07-05 スリーエム イノベイティブ プロパティズ カンパニー Method for enhancing signal detection of cell wall constituents of cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11183479A (en) * 1997-10-16 1999-07-09 Fuji Electric Co Ltd Sensor for measuring solution and method for measuring solution component
JP2002122600A (en) * 2000-10-12 2002-04-26 Nippon Laser & Electronics Lab Sensor chip for biosensor
WO2005038457A1 (en) * 2003-10-15 2005-04-28 Daiichi Pure Chemicals Co., Ltd. Method of separating and assaying adiponectin multimer
JP2007518074A (en) * 2003-12-30 2007-07-05 スリーエム イノベイティブ プロパティズ カンパニー Method for enhancing signal detection of cell wall constituents of cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIYOSHI TAKAYAMA ET AL.: "Development of high speed western-blot system micro-tip type", REPORTS OF SAITAMA INDUSTRIAL TECHNOLOGY CENTER, vol. 7, 2 July 2009 (2009-07-02), pages 119 - 122 *

Also Published As

Publication number Publication date
JP5685777B2 (en) 2015-03-18
JPWO2011135692A1 (en) 2013-07-18

Similar Documents

Publication Publication Date Title
JP5595382B2 (en) Apparatus and method for detecting specimen in saliva
US11198118B2 (en) Integrated modular unit containing one or more analyte concentrator-microreactor devices to be coupled to a cartridge-cassette and methods of operation
JP2020098202A (en) Assay system and cartridge device
RU2011112853A (en) ROTATING MAGNETIC FIELD FOR IMPROVED DETECTION IN THE ANALYSIS OF CLUSTERS
WO2008053822A1 (en) Method of detecting specific bond reaction of molecule by single molecule fluorometry
Luo et al. Application of solid-state nanopore in protein detection
US8940495B2 (en) Rapid and sensitive method for quantitative determination of the level of heparin—PF4 complex induced immunoglobulin antibodies
Manta et al. Tools to evaluate the conformation of protein products
Haab et al. High-throughput studies of protein glycoforms using antibody–lectin sandwich arrays
JP2007333612A (en) Surface plasmon resonance biosensor, and cell response measuring device and measuring method
Bossi et al. Capillary electrophoresis coupled to biosensor detection
Pedersen et al. In-solution IgG titer determination in fermentation broth using affibodies and flow-induced dispersion analysis
Jarrige et al. A fast intraoperative PTH point-of-care assay on the Philips handheld magnotech system
Pinto et al. Knowing more from less: miniaturization of ligand-binding assays and electrophoresis as new paradigms for at-line monitoring and control of mammalian cell bioprocesses
BR112022020895A2 (en) METHOD FOR DETECTING AN ANALYTE, METHOD FOR DETECTING A PEPTIDE ANALYTE AND SYSTEM FOR PERFORMING A TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY METHOD
KR100968334B1 (en) Method for detecting multiple analytes for use in a biosensor
JP5685777B2 (en) Virtual western blotting system
US20050147720A1 (en) Method of analyzing protein
JP2007147539A (en) Immunoassay method, protein analyzer, and micro flow cell for immunoassay
CA2985712C (en) Biosensor device for sensing an analyte in a sample
JP4632156B2 (en) Analysis method using fluorescence depolarization
JP7375940B2 (en) Target analysis device, target analysis method, and target analysis system
JPH1090168A (en) Quantitative and/or qualitative analysis method of atom or molecule
Boschis et al. Smart micro-sensing: Antibodies and aptamer-based micro-ELISA as performing offline/on line tool for allergens and mycotoxins detection in foods
US20220404350A1 (en) Method for determination of aggregates

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10850710

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2012512589

Country of ref document: JP

122 Ep: pct application non-entry in european phase

Ref document number: 10850710

Country of ref document: EP

Kind code of ref document: A1